Biotransformation of colchicinoid compounds

ABSTRACT

The present invention relates to a biotransformation process, effected by means of selected microbial strains, for the preparation of 3-O-glycosyl derivatives of colchicinoid compounds.

This application is a national stage entry of PCT/EP04/05074, filed May12, 2004, the disclosure of which is incorporated by reference.

The present invention relates to a biotransformation process, effectedby means of selected microbial strains, for the preparation of3-O-glycosyl derivatives of colchicinoid compounds.

In particular, the process of the present invention providescolchicinoid compounds glycosylated exclusively at C-3 of the aromaticring A, starting from colchicine, thiocolchicine or the derivativesthereof. The process, resulting in high productivity and purity of theobtained product, is based on an early activation of the glycosylatingenzyme, determined by a demethylated colchicine intermediate.Preferably, the process of the invention consists of a multiplefeed-batch fermentation process, comprising the fractionated feeding ofa nitrogen source, a carbon source and the substrate to be transformed.

The colchicinoid compounds glycosylated at C-3 of the benzene ring areof remarkable pharmacological importance in view of their higheffectiveness and as intermediates for the preparation of newmedicaments.

In particular, thiocolchicoside (3-O-glucosylthiocolchicine) is anattractive and interesting active ingredient in the pharmaceuticalfield, mainly in the therapy of diseases of the muscle-skeletal system,and as starting materials for the preparation of novel antitumor,immunosuppressive, antipsoriasis and antiinflammatory medicaments.

WO 98/15642 discloses a process for the production of colchicinoidglycosyl-derivatives in high conversion yields (up to 90%), startingfrom colchicinoid compounds such as colchicine, thiocolchicine andderivatives thereof, with an initial concentration up to 1 g/l. Theprocess is based on a preliminary, regioselective demethylation step ofthe C-3 methoxy group bound to the aromatic ring of the colchicinoid,and a subsequent glycosylation of the demethyl derivative at the samemolecular site.

The process of the present invention, while maintaining the same highconversion yield of the former, allows the transformation ofconsiderably higher amount of total substrate, thus improving both thetotal productivity, expressed as the total amount of product obtainedper liter per batch, and the specific productivity, expressed as theamount of product obtained per liter per time unit (i.e: hour) duringthe biotransformation process.

The process of the invention is characterised by the activationmechanism of the glycosylating step, based on the induction of thespecific enzyme. In fact, it has been surprisingly found that the enzymeinvolved in the glycosylation step is efficiently induced bydemethylated colchicinoids such as 3-O-demethylcolchicine (DMC) or3-O-demethylthiocolchicine (DMTC). An addition of small amounts (200-600mg/l) of the 3-demethyl derivative in the early stages of thebiotransformation process, or preferably in the preliminary seedculture, is useful for an early activation of the glycosylating system.In such condition, the demethylated intermediate, step-by-step releasedby the demethylating enzyme, can be efficiently converted into the finalglycosyl-derivative, and the biotransformation can proceed very fast,during the first 15-18 hours of fermentation. Moreover there is nosignificant accumulation of the intermediate, that means no inhibitionof the demethylating activity and high conversion rate. Thebiotransformation is completed after 18-21 hours and is thereforeconsiderably faster than that of the known process (26-28 hours). Theconversion yield remains very high, from 80% to 100%, usually about90-95%, but with a considerable increase of total transformed substrateand, consequently, of final product.

The invention therefore provides a process for the preparation of3-O-glycosylcolchicinoid compounds of formula (I),

wherein R₁ is an O-glycosyde residue, R₂ is hydrogen or C₁-C₇ acyl, R₃is C₁-C₆ alkoxy or C₁-C₆ thioalkyl, comprising the biotransformation ofcompounds in which R₁ is OH or methoxy by means of Bacillus megaterium,characterised in that demethylated colchicinoids are used to induce theglycosylating enzyme system.

R₁ is preferably an O-glucoside residue.

A preferred embodiment of the process of the invention, consists in amultiple, fractionated feeding of the substrate, in combination with anitrogen and a carbon source, like peptone and glucose. This condition,by assuring a stable growth rate of the microbial culture, allows arelevant increase of the total substrate added to the fermentation batch(2-4 g/l, instead of 1 g/l) and consequently of the productivity of theprocess (up to 2.5-5.2 g/l of glycosylated product per single batch,instead of 1-1.2 g/l), without any loss of conversion efficiency andrisk of toxic effects on the bacterial culture, due to a considerableaccumulation of the untransformed substrate.

The present invention is also advantageous in view of the reduced timeof the process, resulting in a high viability of the culture and alimited cell lysis, with reduced cell debris formation and considerableadvantages for the downstream processing and the recovery of theproduct. This advantage can be further improved by the use of absorptionresins, like XAD 1180 (Rohm & Haas) or HP 21 (Mitsubishi), useful for aselective absorption of the colchicinoid compounds and an efficientproduct recovery from the fermentation broth.

A further advantage concerns the possibility of a semi-continuousprocess, by recovering the main fraction (75%-90%) of the finalfermentation broth, containing the product, adding new fermentationmedium to the remaining part of the broth in the bioreactor, andstarting with a new batch. This approach can be extended to repeatedfermentation steps, with a proportional increase of the totalproductivity.

Moreover the constant regioselectivity of the catalysis assures, inaddition to the remarkable production yields, a high quality of theresulting product, allowing a purity ≧99% with a simple downstreamprocessing and a reduced incidence of the step of purification andrecovery of the product. This aspect implies further advantages in termsof solvent limitation and high environmental compatibility of theprocess.

The Bacillus megaterium microorganisms usable in the present inventionare the same described in WO 98/15642, herein incorporated forreference.

They can be selected on different agar media containing an organicnitrogen source (peptones, yeast extracts, meat extracts, asparagine,etc.), a carbon source (glycerin, starch, maltose, glucose, etc.), withpH 5 to 8, preferably 6-7. The incubation temperature ranges from 20° to45° C., preferably 28°-40° C.

The culture media used for the conservation of the culture are typicalmicrobiological substrates, containing organic nitrogen sources(peptones, yeast extracts, tryptone, meat extracts, etc.), a carbonsource (glucose, maltose, glycerin, etc.), at pH 5 to 8, preferably 6-7.The incubation temperature ranges from 20° to 45° C., preferably 28°-40°C.

The selected microorganisms can be assayed for the capability of growingin submerged culture, in the presence of colchicinoid compounds, addedas described in the present invention, and of transforming the latterinto the corresponding 3-glycosyl derivatives.

Said assays were carried out in 100 ml flasks containing 20 ml of liquidmedium, with different medium formulations, comprising one or moreorganic nitrogen sources (yeast extracts, peptones, tryptone, caseinhydrolysates, meat extract, corn-step liquor, etc.), one or more carbonsources (glucose, glycerol, starch, saccharose, etc.), inorganicphosphorous and nitrogen sources, and inorganic salts of various ions(K+, Na+, Mg++, Ca++, Fe++, Mn++, etc.).

The culture samples can optionally be subjected to mutagenic treatments,by means of the conventional mutagenesis techniques (irradiation with UVrays, etc.) to induce mutants having a specific bioconversion activitywhich can be evaluated with the same procedure as above.

The capability of the selected bacteria of transforming into therespective 3-glycosyl derivatives colchicinoid substrates, added to theculture broth in repeated fractions, together with a carbon and anitrogen source, has been confirmed by means of bioconversion assays inflasks, in a 300 ml scale, containing different medium formulations,comprising one or more organic nitrogen sources (yeast extracts,peptones, tryptone, casein hydrolysates, meat extract, corn-step liquor,etc.), one or more carbon sources (glucose, glycerol, starch,saccharose, etc.), inorganic phosphorous and nitrogen sources, andinorganic salts of various ions (K+, Na+, Mg++, Ca++, Fe++, Mn++, NH4+,etc.).

Bacterial growth and biotransformation are supported by one or moreorganic nitrogen sources, preferably meat extract, peptone, tryptone,casein hydrolysates, corn-steep liquor, etc. Carbon sources useful forgrowth and biotransformation are glucose, fructose, saccharose,glycerol, malt extract, etc., preferably glucose, fructose and glycerol.The culture medium contains moreover inorganic phosphorous sources andsalts of K+, Na+, Mg++, Mn++, NH4+, etc.

The carbon source useful for in process feeding are preferably glucoseand fructose. The nitrogen source for in process feeding is preferablyan organic source, like peptone, casein hydrolysate, tryptone, or aninorganic one, like ammonium sulphate, or a combination of both.

Preliminary experiments have shown that the introduction, before thebiotransformation step, of a submerged seed culture, containing a mediumformulation similar to that of the production medium, results in a veryhomogeneous and highly active microbial population at the beginning ofthe biotransformation.

Additional experiments have been performed using different fermentationparameters, like amount, time and modality of substrate addition,feeding ratio, incubation time, inoculum ratio, etc.

An early addition of a demethyl colchicinoid as inducer, at thebeginning of the biotransformation step or, better, during thepreliminary seed culture, can considerably increase the conversionefficiency and the total productivity of the process.

Further increase can be achieved by feeding the substrate to beconverted in more fractions during the process, in combination with acarbon source and a nitrogen source.

A typical procedure, combining the parameters described above, cantherefore be based on the following schedule:

-   -   a preliminary addition of a 3-O-demethyl colchicinoid (200-600        mg/l, preferably 300-500 mg/l) in the seed culture;    -   addition of aliquots (300-800 mg/l, preferably 500-600 mg/l) of        the colchicinoid substrate to be converted, at the beginning and        every 1-3 hours, preferably 1.5-2.5 hours, during the first        14-18 hours, preferably 15-16 hours of biotransformation;    -   addition, at each time of the substrate feeding, of a solution        containing the following raw materials:    -   a) peptone, or tryptone, or caseine hydrolysate, at a final        concentration between 2 and 4 g/l, preferably 2.5-3.5 g/l (also        in combination with ammonium sulphate 1-3 g/l, preferably        1.5-2.5 g/l);    -   b) glucose or fructose, at a final concentration between 5 and        15 g/l, preferably 8-12 g/l.

Considering a total fermentation time of 20 hours, an initial substrateconcentration of 500 g/l, an in-process feeding interval of 2 hours anda total number of 7 feeding steps (last substrate addition at 14thfermentation hour), a total substrate amount of 4 g/l (instead of 1 g/l,as described in WO 98/15642) will be added to the culture.

The biotransformation can be performed at 25°-35° C., preferably at28°-32° C., at pH between 4 and 8, preferably 5-7, in stirred flasks ona rotary shaker.

The biotransformation of the invention can be scaled up to fermentationtank level, keeping the culture conditions unchanged, in particular asfar as culture medium, temperature and processing times are concerned.In order to obtain good growths, adequate levels of stirring-aerationare important, in particular aeration levels of 1-2 liters of air perliter of culture per minute (vvm), preferably of 1.4-1.8 vvm, arerequired. In such condition the process is very fast and after 20-21hours the biotransformation is completed.

The product is extracellular and can be extracted from the culture brothafter separation of the biomass from the liquid fraction bycentrifugation and recovery of the supernatant, or microfiltration andrecovery of the permeate. The culture can be treated with alcohols, inview of an optimum recovery of the product.

The purification can be performed by chromatographic techniques,liquid-liquid extraction with alcohols and lipophylic organic solventsand crystallization, as described in WO 98/154621.

The following examples disclose the invention in further detail.

EXAMPLE 1

An aliquot of frozen culture of Bacillus megaterium is utilized for theinoculum of seed cultures (i.e.: preculture) in 1000 ml Erlenmeyerflask, containing 250 ml of medium SF2 (Table), added with3-O-demethylthiocolchicine to a 0.4 g/l final concentration. Saidcultures are incubated overnight at 30° C., on a rotary shaker, at 250rpm. After incubation, 500 ml of preculture are transferred in sterileinto a 14 l fermenter, containing 9.5 l of fresh medium SF2 (see Table1), added with thiocolchicine to a 0.5 g/l final concentration. Thefermentation is carried out at 30° C., keeping suitable levels ofstirring-aeration (stirring up to 900 rpm; aeration 1 to 1.8 vvm,depending on the culture growth). Every 2 hours thiocolchicine (0.5 g/lfinal concentration), peptone (2 g/l), ammonium sulphate (2 g/l) andglucose (10 g/l) are added to the culture, during the first 14 hours offermentation. Before each addition (i.e.: every 2 hours) samples fromthe culture broths are taken and subjected to the following analysis:

-   -   Growth level, as optical density (OD) at 600 nm;    -   Sterility and purity analysis of the strain on LB Agar;    -   Microscope morphology (Gram stain);    -   Analysis of the thiocolchicoside content, by TLC and HPLC.

TLC analysis is performed on silica gel, with an acetone:ethylacetate:water 5:4:1 eluent system. For the HPLC analysis, 1 ml fractionsof culture broths are added with 9 ml of methanol and centrifuged at13,000 rpm for 2 minutes. The content in thiocolchicoside of thesupernatant is analysed by reverse phase HPLC, with isocratic elution,by means of the water:acetonitrile 80:20 eluent system. The HPLCanalysis proves that the conversion of thiocolchicine intothiocolchicoside starts very early and is almost completed after 20hours. A total amount of 4 g/l of thiocolchicine are transformed into5.2 g/l of thiocolchicoside, with a 96% conversion yield and a specificproductivity of 0.26 g/l·h of glucosylated colchicinoid.

EXAMPLE 2

The final culture broth from the fermentation (total volume: about 10l), containing about 52 g of thiocolchicoside as determined by HPLCanalysis, is subjected to cross-flow microfiltration on a 0.22 μmceramic cartridge, to separate the cells from the broth. The permeate isabsorbed on a column filled with a XAD 1180 (Rohm and Haas) absorptionresin. After washing with water, the product is eluted with methanol.The methanol eluate is concentrated to dryness under vacuum, thenredissolved in methanol. After extraction with methylene chloride, thealcohol fraction is concentrated to dryness and redissolved in anethanol-methylene chloride, 1:1 mixture. After clarification with silicagel, the solution is concentrated under vacuum; methylene chloride isthen substituted with ethanol. The resulting suspension is concentratedand left to crystallize. A second crystallization with ethanol iscarried out after further redissolution steps of the solid inethanol-chloroform mixtures and clarification on silica gel. A totalamount of 49.9 g of product are obtained after purification, with apurification yield of 96% and a purity of 99.5%.

The resulting product, analysed by HPLC, C-NMR, H-NMR and mass spectrum,turns out to be the same as the thiocolchicoside standard.

COMPARATIVE EXAMPLE 3

The procedure described in Example 1 is repeated, but thiocolchicine istotally added in a single fraction, at a final concentration of 4 g/l,at the beginning of fermentation. The resulting growth is very poor andis practically blocked after a few hours of incubation. Evident celllysis is detected by microscopic analysis. No significantbiotransformation is shown by TLC and HPLC analysis.

COMPARATIVE EXAMPLE 4

The procedure described in Example 1 is repeated, but without anyaddition of 3-O-demethylthiocolchicine in the preliminary seed culture(no enzyme induction). Biotransformation results slower than that as inExample 1, and is stopped after 28 hours, with a final conversion yieldof 61%, a total productivity of 3.3 g/l and a specific productivity of0.118 g/l·h of thiocolchicoside.

COMPARATIVE EXAMPLE 5 Corresponding to WO 98/15462

The procedure described in Example 4 is repeated, but thiocolchicine istotally added in a single fraction, at a final concentration of 1 g/l,at the beginning of fermentation, using fermentation medium ST asdescribed in WO98/15462. Biotransformation results slower than that asin example 1, and is stopped after 28 hours, with a final conversionyield of 90%, a total productivity of 1.22 g/l and a specificproductivity of 0.044 g/l·h of thiocolchicoside.

The following table shows a comparison in terms of total productivity,specific productivity and conversion yield of thiocolchicine intothiocolchicoside, between the method of the present invention (A) andthat of WO098/15642 (B).

Productivity (total) Productivity Conversion yield g/l (specific) g/l %A* 5.22 0.261 96 B* 1.22 0.044 90 *substrate (thiocolchicine) added; 4g/l (A); 1 g/l (B)

TABLE 1 Formulation of the culture media 1) LB-Agar (Sterilization: 121°C. × 20′)-pH 7 Triptone 10 g/l Yeast extract 5 g/l NaCl 10 g/l Agar Agar15 g/l 2) Broth SF2 (Sterilization: 121° C. × 20′)-pH 7 Glucose 40 g/lPeptone 20 g/l Yeast extract 5 g/l NaCl 3 g/l (NH₄)₂SO₄ 3 g/l K₂HPO₄ 8g/l KH₂PO₄ 3 g/l MgSO₄•7H₂0 0.5 g/l

1. A process for preparing a 3-O-glycosylcolchicinoid compound offormula I:

wherein R₁ is a O-glycoside residue, R₂ is hydrogen or C₁-C₇ acyl, andR₃ is C₁-C₆ alkoxy or C₁-C₆ thioalkyl, comprising: a) contactingBacillus megaterium with a demethylcochicinoid compound selected fromthe group consisting of 3-O-demethylcholchicine (DMC) and3-O-demethylthiocholchicine (DMTC), wherein the demethylcochicinoidcompound induces a glycosylating enzyme of the Bacillus megaterium, toobtain a preliminary seed culture; and b) fermenting a mixturecomprising a carbon source, a nitrogen source and a compound of FormulaII:

wherein R₂ is hydrogen or C₁-C₇ acyl and R₃ is C₁-C₆ alkoxy or C₁-C₆thioalkyl, with the preliminary seed culture to form a broth, whereinmultiple fractions of the mixture are added to the broth during thefermentation.
 2. The process according to claim 1, wherein theconcentration of the demethylated colchicinoid compound is from 200 to600 mg/l per fraction added.
 3. The process according to claim 1,wherein the nitrogen source is peptone and the carbon source is glucose.4. The process according to claim 1, wherein the final concentration ofthe compound of formula II added to the fermentation batch is 2-4 g/l.5. The process according to claim 1, wherein the compound of formula Iis recovered by contact with an absorption resin.
 6. The processaccording to claim 1, wherein the process is carried out in asemi-continuous manner comprising recovering a fraction comprising75-90% of the fermentation broth containing the compound of formula Iand adding additional amounts of the mixture to the remaining portion ofthe fermentation broth.
 7. The process according to claim 1, wherein thefractions of the mixture comprising the carbon source, nitrogen sourceand the compound of formula II are added at the beginning of thefermentation step and every 1-3 hours thereafter during the first 14-18hours of the fermentation process; wherein each fraction comprises thecompound of formula II in a concentration of 300-800 mg/ml; the nitrogensource is ammonium sulfate at a concentration of 1-3 g/l in combinationwith an additional nitrogen source selected from the group consisting ofpeptone, tryptone and caseine hydrolysate wherein final concentration ofthe additional nitrogen source is 2-4 g/l; and the carbon source isselected from the group consisting of glucose and fructose, wherein thecarbon source has a final concentration of 5-15 g/l.
 8. The processaccording to claim 1, wherein the fermentation is carried out at 25° to35° C. and a pH of between 4 and
 8. 9. The process according to claim 1,wherein the broth is aerated at a level of 1-2 liters of air per literof broth per minute.